Lingulodinium polyedrum is a unicellular marine organism which belongs to the dinoflagellate group of algae. Its genome is among the largest found in any species on this planet, estimated to contain around 165 billion DNA base pairs – roughly fifty times larger than the size of the human genome. Encased in magnificent polyhedral shells, these bioluminescent algae became important organisms to study biological rhythms. Each Lingulodinium polyedrum cell contains not one but at least two internal clocks which keep track of time by oscillating at a frequency of approximately 24 hours. Algae maintained in continuous light for weeks continue to emit a bluish-green glow at what they perceive as night-time and swim up to the water surface during day-time hours – despite the absence of any external time cues. When I began studying how nutrients affect the circadian rhythms of these algae as a student at the University of Munich, I marveled at the intricacy and beauty of these complex time-keeping mechanisms that had evolved over hundreds of millions of years.

I was prompted to revisit the role of Beauty in biology while reading a masterpiece of scientific writing, “Dreams of a Final Theory” by the Nobel laureate Steven Weinberg in which he describes how the search for Beauty has guided him and many fellow theoretical physicists to search for an ultimate theory of the fundamental forces of nature. Weinberg explains that it is quite difficult to precisely define what constitutes Beauty in physics but a physicist would nevertheless recognize it when she sees it.Over the course of a quarter of a century, I have worked in a variety of biological fields, from these initial experiments in marine algae to how stem cells help build human blood vessels and how mitochondria in a cell fragment and reconnect as cells divide. Each project required its own set of research methods and techniques, each project came with its own failures and successes. But with each project, my sense of awe for the beauty of nature has grown. Evolution has bestowed this planet with such an amazing diversity of life-forms and biological mechanisms, allowing organisms to cope with the unique challenges that they face in their respective habitats. But it is only recently that I have become aware of the fact that my sense of biological beauty was a post hoc phenomenon: Beauty was what I perceived after reviewing the experimental findings; I was not guided by a quest for beauty while designing experiments. In fact, I would have been worried that such an approach might bias the design and interpretation of experiments. Might a desire for seeing Beauty in cell biology lead one to consciously or subconsciously discard results that might seem too messy?

One such key characteristic of a beautiful scientific theory is the simplicity of the underlying concepts. According to Weinberg, Einstein’s theory of gravitation is described in fourteen equations whereas Newton’s theory can be expressed in three. Despite the appearance of greater complexity in Einstein’s theory, Weinberg finds it more beautiful than Newton’s theory because the Einsteinian approach rests on one elegant central principle – the equivalence of gravitation and inertia. Weinberg’s second characteristic for beautiful scientific theories is their inevitability. Every major aspect of the theory seems so perfect that it cannot be tweaked or improved on. Any attempt to significantly modify Einstein’s theory of general relativity would lead to undermining its fundamental concepts, just like any attempts to move around parts of Raphael’s Holy Family would weaken the whole painting.

Can similar principles be applied to biology? I realized that when I give examples of beauty in biology, I focus on the complexity and diversity of life, not its simplicity or inevitability. Perhaps this is due to the fact that Weinberg was describing the search of fundamental laws of physics, laws which would explain the basis of all matter and energy – our universe. As cell biologists, we work several orders of magnitude removed from these fundamental laws. Our building blocks are organic molecules such as proteins and sugars. We find little evidence of inevitability in the molecular pathways we study – cells have an extraordinary ability to adapt. Mutations in genes or derangement in molecular signaling can often be compensated by alternate cellular pathways.

This also points to a fundamental difference in our approaches to the world. Physicists searching for the fundamental laws of nature balance the development of fundamental theories whereas biology in its current form has primarily become an experimental discipline. The latest technological developments in DNA and RNA sequencing, genome editing, optogenetics and high resolution imaging are allowing us to amass unimaginable quantities of experimental data. In fact, the development of technologies often drives the design of experiments. The availability of a genetically engineered mouse model that allows us to track the fate of individual cells that express fluorescent proteins, for example, will give rise to numerous experiments to study cell fate in various disease models and organs. Much of the current biomedical research funding focuses on studying organisms that provide technical convenience such as genetically engineered mice or fulfill a societal goal such as curing human disease.

Uncovering fundamental concepts in biology requires comparative studies across biology and substantial investments in research involving a plethora of other species. In 1990, the National Institutes of Health (NIH – the primary government funding source for biomedical research in the United States) designated a handful of species as model organisms to study human disease, including mice, rats, zebrafish and fruit flies. A recent analysis of the species studied in scientific publications showed that in 1960, roughly half the papers studied what would subsequently be classified as model organisms whereas the other half of papers studied additional species. By 2010, over 80% of the scientific papers were now being published on model organisms and only 20% were devoted to other species, thus marking a significant dwindling of broader research goals in biology. More importantly, even among the model organisms, there has been a clear culling of research priorities with a disproportionately large growth in funding and publications for studies using mice. Thousands of scientific papers are published every month on the cell signaling pathways and molecular biology in mouse and human cells whereas only a minuscule fraction of research resources are devoted to studying signaling pathways in algae.

The question of whether or not biologists should be guided by conceptual Beauty leads us to the even more pressing question of whether we need to broaden biological research. If we want to mirror the dizzying success of fundamental physics during the past century and similarly advance fundamental biology, then we need substantially step-up investments in fundamental biological research that is not constrained by medical goals.

Billions of cells die each day in the human body in a process called “apoptosis” or “programmed cell death”. When cells encounter stress such as inflammation, toxins or pollutants, they initiate an internal repair program which gets rid of the damaged proteins and DNA molecules. But if the damage exceeds their capacity for repair then cells are forced to activate the apoptosis program. Apoptotic cells do not suddenly die and vanish, instead they execute a well-coordinated series of molecular and cellular signals which result in a gradual disintegration of the cell over a period of several hours.

The remains of an apoptotic cell are being engulfed and ingested by a phagocytic white blood cell. Image via National Library of Medicine.

What happens to the cellular debris that is generated when a cell dies via apoptosis? It consists of fragmented cellular compartments, proteins, fat molecules that are released from the cellular corpse. This “trash” could cause even more damage to neighboring cells because it exposes them to molecules that normally reside inside a cell and could trigger harmful reactions on the outside. Other cells therefore have to clean up the mess as soon as possible. Macrophages are cells which act as professional garbage collectors and patrol our tissues, on the look-out for dead cells and cellular debris. The remains of the apoptotic cell act as an “Eat me!” signal to which macrophages respond by engulfing and gobbling up the debris (“phagocytosis“) before it can cause any further harm. Macrophages aren’t always around to clean up the debris which is why other cells such as fibroblasts or epithelial cells can act as non-professional phagocytes and also ingest the dead cell’s remains. Nobody likes to be surrounded by trash.

Clearance of apoptotic cells and their remains is thus crucial to maintain the health and function of a tissue. Conversely, if phagocytosis is inhibited or prevented, then the lingering debris can activate inflammatory signals and cause disease. Multiple autoimmune diseases, lung diseases and even neurologic diseases such as Alzheimer’s disease are associated with reduced clearance. The cause and effect relationship is not always clear because these diseases can promote cell death. Are the diseases just killing so many cells that the phagocytosis capacity is overwhelmed, does the debris actually promote the diseased state, or is it a bit of both, resulting in a vicious cycle of apoptotic debris resulting in more cell death and more trash buildup? Researchers are currently investigating whether specifically tweaking phagocytosis could be used as a novel way to treat diseases with impaired clearance of debris.

During the past decade, multiple groups of researchers have come across a fascinating phenomenon by which viruses hijack the phagocytosis process in order to thrive. One of the “Eat Me!” signals for phagocytes is that debris derived from an apoptotic cell is coated by a membrane enriched with phosphatidylserines which are negatively charged molecules. Phosphatidylserines are present in all cells but they are usually tucked away on the inside of cells and are not seen by other cells. When a cell undergoes apoptosis, phosphatidylserines are flipped inside out. When particles or cell fragments present high levels of phosphatidylserines on their outer membranes then a phagocyte knows that it is encountering the remains of a formerly functioning cell that needs to be cleared by phagocytosis.

However, it turns out that not all membranes rich in phosphatidylserines are remains of apoptotic cells. Recent research studies suggest that certain viruses invade cells, replicate within the cell and when they exit their diseased host cell, they cloak themselves in membranes rich in phosphatidylserines. How the viruses precisely appropriate the phosphatidylserines of a cell that is not yet apoptotic and then adorn their viral membranes with the cell’s “Eat Me!” signal is not yet fully understood and a very exciting area of research at the interface of virology, immunology and the biology of cell death.

What happens when the newly synthesized viral particles leave the infected cell? Because these viral particles are coated in phosphatidylserine, professional phagocytes such as macrophages or non-professional phagocytes such as fibroblasts or epithelial cells will assume they are encountering phosphatidylserine-rich dead cell debris and ingest it in their roles as diligent garbage collectors. This ingestion of the viral particles has at least two great benefits for the virus: First and foremost, it allows the virus entry into a new host cell which it can then convert into another virus-producing factory. Entering cells usually requires specific receptors by which viruses gain access to selected cell types. This is why many viruses can only infect certain cell types because not all cells have the receptors that allow for viral entry. However, when viruses hijack the apoptotic debris phagocytosis mechanism then the phagocytic cell is “inviting” the viral particle inside, assuming that it is just dead debris. But there is perhaps an even more insidious advantage for the virus. During clearance of apoptotic cells, certain immune pathways are suppressed by the phagocytes in order to pre-emptively dampen excessive inflammation that might be caused by the debris. It is therefore possible that by pretending to be fragments of dead cells, viruses coated with phosphatidylserines may also suppress the immune response of the infected host, thus evading detection and destruction by the immune systems.

Viruses for which this process of apoptotic mimicry has been described include the deadly Ebola virus or the Dengue virus, each using its own mechanism to create its fake mask of death. The Ebola virus buds directly from the fat-rich outer membrane of the infected host cell in the form of elongated, thread-like particles coated with the cell’s phosphatidylserines. The Dengue virus, on the other hand, is synthesized and packaged inside the cell and appears to purloin the cell’s phosphatidylserines during its synthesis long before it even reaches the cell’s outer membrane. As of now, it appears that viruses from at least nine distinct families of viruses use the apoptotic mimicry strategy but the research on apoptotic mimicry is still fairly new and it is likely that scientists will discover many more viruses which rely on this and similar evolutionary strategies to evade the infected host’s immune response and spread throughout the body.

Uncovering the phenomenon of apoptotic mimicry gives new hope in the battle against viruses for which we have few targeted treatments. In order to develop feasible therapies, it is important to precisely understand the molecular mechanisms by which the hijacking occurs. One cannot block all apoptotic clearance in the body because that would have disastrous consequences due to the buildup of legitimate apoptotic debris that needs to be cleared. However, once scientists understand how viruses concentrate phosphatidylserines or other “Eat Me!” signals in their membranes, it may be possible to specifically uncloak these renegade viruses without compromising the much needed clearance of conventional cell debris.

Murder your darlings. The British writer Sir Arthur Quiller Crouch shared this piece of writerly wisdom when he gave his inaugural lecture series at Cambridge, asking writers to consider deleting words, phrases or even paragraphs that are especially dear to them. The minute writers fall in love with what they write, they are bound to lose their objectivity and may not be able to judge how their choice of words will be perceived by the reader. But writers aren’t the only ones who can fall prey to the Pygmalion syndrome. Scientists often find themselves in a similar situation when they develop “pet” or “darling” hypotheses.

How do scientists decide when it is time to murder their darling hypotheses? The simple answer is that scientists ought to give up scientific hypotheses once the experimental data is unable to support them, no matter how “darling” they are. However, the problem with scientific hypotheses is that they aren’t just generated based on subjective whims. A scientific hypothesis is usually put forward after analyzing substantial amounts of experimental data. The better a hypothesis is at explaining the existing data, the more “darling” it becomes. Therefore, scientists are reluctant to discard a hypothesis because of just one piece of experimental data that contradicts it.

In addition to experimental data, a number of additional factors can also play a major role in determining whether scientists will either discard or uphold their darling scientific hypotheses. Some scientific careers are built on specific scientific hypotheses which set apart certain scientists from competing rival groups. Research grants, which are essential to the survival of a scientific laboratory by providing salary funds for the senior researchers as well as the junior trainees and research staff, are written in a hypothesis-focused manner, outlining experiments that will lead to the acceptance or rejection of selected scientific hypotheses. Well written research grants always consider the possibility that the core hypothesis may be rejected based on the future experimental data. But if the hypothesis has to be rejected then the scientist has to explain the discrepancies between the preferred hypothesis that is now falling in disrepute and all the preliminary data that had led her to formulate the initial hypothesis. Such discrepancies could endanger the renewal of the grant funding and the future of the laboratory. Last but not least, it is very difficult to publish a scholarly paper describing a rejected scientific hypothesis without providing an in-depth mechanistic explanation for why the hypothesis was wrong and proposing alternate hypotheses.

For example, it is quite reasonable for a cell biologist to formulate the hypothesis that protein A improves the survival of neurons by activating pathway X based on prior scientific studies which have shown that protein A is an activator of pathway X in neurons and other studies which prove that pathway X improves cell survival in skin cells. If the data supports the hypothesis, publishing this result is fairly straightforward because it conforms to the general expectations. However, if the data does not support this hypothesis then the scientist has to explain why. Is it because protein A did not activate pathway X in her experiments? Is it because in pathway X functions differently in neurons than in skin cells? Is it because neurons and skin cells have a different threshold for survival? Experimental results that do not conform to the predictions have the potential to uncover exciting new scientific mechanisms but chasing down these alternate explanations requires a lot of time and resources which are becoming increasingly scarce. Therefore, it shouldn’t come as a surprise that some scientists may consciously or subconsciously ignore selected pieces of experimental data which contradict their darling hypotheses.

Let us move from these hypothetical situations to the real world of laboratories. There is surprisingly little data on how and when scientists reject hypotheses, but John Fugelsang and Kevin Dunbar at Dartmouth conducted a rather unique study “Theory and data interactions of the scientific mind: Evidence from the molecular and the cognitive laboratory” in 2004 in which they researched researchers. They sat in at scientific laboratory meetings of three renowned molecular biology laboratories at carefully recorded how scientists presented their laboratory data and how they would handle results which contradicted their predictions based on their hypotheses and models.

In their final analysis, Fugelsang and Dunbar included 417 scientific results that were presented at the meetings of which roughly half (223 out of 417) were not consistent with the predictions. Only 12% of these inconsistencies lead to change of the scientific model (and thus a revision of hypotheses). In the vast majority of the cases, the laboratories decided to follow up the studies by repeating and modifying the experimental protocols, thinking that the fault did not lie with the hypotheses but instead with the manner how the experiment was conducted. In the follow up experiments, 84 of the inconsistent findings could be replicated and this in turn resulted in a gradual modification of the underlying models and hypotheses in the majority of the cases. However, even when the inconsistent results were replicated, only 61% of the models were revised which means that 39% of the cases did not lead to any significant changes.

The study did not provide much information on the long-term fate of the hypotheses and models and we obviously cannot generalize the results of three molecular biology laboratory meetings at one university to the whole scientific enterprise. Also, Fugelsang and Dunbar’s study did not have a large enough sample size to clearly identify the reasons why some scientists were willing to revise their models and others weren’t. Was it because of varying complexity of experiments and models? Was it because of the approach of the individuals who conducted the experiments or the laboratory heads? I wish there were more studies like this because it would help us understand the scientific process better and maybe improve the quality of scientific research if we learned how different scientists handle inconsistent results.

In my own experience, I have also struggled with results which defied my scientific hypotheses. In 2002, we found that stem cells in human fat tissue could help grow new blood vessels. Yes, you could obtain fat from a liposuction performed by a plastic surgeon and inject these fat-derived stem cells into animal models of low blood flow in the legs. Within a week or two, the injected cells helped restore the blood flow to near normal levels! The simplest hypothesis was that the stem cells converted into endothelial cells, the cell type which forms the lining of blood vessels. However, after several months of experiments, I found no consistent evidence of fat-derived stem cells transforming into endothelial cells. We ended up publishing a paper which proposed an alternative explanation that the stem cells were releasing growth factors that helped grow blood vessels. But this explanation was not as satisfying as I had hoped. It did not account for the fact that the stem cells had aligned themselves alongside blood vessel structures and behaved like blood vessel cells.

Even though I “murdered” my darling hypothesis of fat –derived stem cells converting into blood vessel endothelial cells at the time, I did not “bury” the hypothesis. It kept ruminating in the back of my mind until roughly one decade later when we were again studying how stem cells were improving blood vessel growth. The difference was that this time, I had access to a live-imaging confocal laser microscope which allowed us to take images of cells labeled with red and green fluorescent dyes over long periods of time. Below, you can see a video of human bone marrow mesenchymal stem cells (labeled green) and human endothelial cells (labeled red) observed with the microscope overnight. The short movie compresses images obtained throughout the night and shows that the stem cells indeed do not convert into endothelial cells. Instead, they form a scaffold and guide the endothelial cells (red) by allowing them to move alongside the green scaffold and thus construct their network. This work was published in 2013 in the Journal of Molecular and Cellular Cardiology, roughly a decade after I had been forced to give up on the initial hypothesis. Back in 2002, I had assumed that the stem cells were turning into blood vessel endothelial cells because they aligned themselves in blood vessel like structures. I had never considered the possibility that they were scaffold for the endothelial cells.

This and other similar experiences have lead me to reformulate the “murder your darlings” commandment to “murder your darling hypotheses but do not bury them”. Instead of repeatedly trying to defend scientific hypotheses that cannot be supported by emerging experimental data, it is better to give up on them. But this does not mean that we should forget and bury those initial hypotheses. With newer technologies, resources or collaborations, we may find ways to explain inconsistent results years later that were not previously available to us. This is why I regularly peruse my cemetery of dead hypotheses on my hard drive to see if there are ways of perhaps resurrecting them, not in their original form but in a modification that I am now able to test.

Back in 2001, when we first began studying how regenerative cells (stem cells or more mature progenitor cells) enhance blood vessel growth, our group as well as many of our colleagues focused on one specific type of blood vessel: arteries. Arteries are responsible for supplying oxygen to all organs and tissues of the body and arteries are more likely to develop gradual plaque build-up (atherosclerosis) than veins or networks of smaller blood vessels (capillaries). Once the amount of plaque in an artery reaches a critical threshold, the oxygenation of the supplied tissues and organs becomes compromised. In addition to this build-up of plaque and gradual decline of organ function, arterial plaques can rupture and cause severe sudden damage such as a heart attack. The conventional approach to treating arterial blockages in the heart was to either perform an open-heart bypass surgery in which blocked arteries were manually bypassed or to place a tube-like “stent” in the blocked artery to restore the oxygen supply. The hope was that injections of regenerative cells would ultimately replace the invasive procedures because the stem cells would convert into blood vessel cells, form healthy new arteries and naturally bypass the blockages in the existing arteries.

As is often the case in biomedical research, this initial approach turned out to be fraught with difficulties. The early animal studies were quite promising and the injected cells appeared to stimulate the growth of blood vessels, but the first clinical trials were less successful. It was very difficult to retain the injected cells in the desired arteries or tissues, and even harder to track the fate of the cells. Which stem cells should be injected? Where should they be injected? How many? Can one obtain enough stem cells from an individual patient so that one could use his or her own cells for the cell therapy? How does one guide the injected cells to the correct location, and then guide the cells to form functional blood vessel structures? Would the stem cells of a patient with chronic diseases such as diabetes or high blood pressure be suitable for therapies, or would such a patient have to rely on stem cells from healthier individuals and thus risk the complication of immune rejection?

The complexity of blood-vessel generation became increasingly apparent, both when studying the biology of stem cells as well as when designing and conducting clinical trials. A large clinical study published in 2013 studied the impact of bone marrow cell injections in heart attack patients and concluded that these injections did not result in any sustained benefit for heart function. Other studies using injections of patients’ own stem cells into their hearts had led to mild improvements in heart function, but none of these clinical studies came close to fulfilling the expectations of cardiovascular patients, physicians and researchers. The upside to these failed expectations was that it forced the researchers in the field of cardiovascular regeneration to rethink their goals and approaches.

One major shift in my own field of interest – the generation of new blood vessels – was to reevaluate the validity of relying on injections of cells. How likely was it that millions of injected cells could organize themselves into functional blood vessels? Injections of cells were convenient for patients because they would not require the surgical implantation of blood vessels, but was this attempt to achieve a convenient therapy undermining its success? An increasing number of laboratories began studying the engineering of blood vessels in the lab by investigating the molecular cues which regulate the assembly of blood vessel networks, identifying molecular scaffolds which would retain stem cells and blood vessel cells and combining various regenerative cell types to build functional blood vessels. This second wave of regenerative vascular medicine is engineering blood vessels which will have to be surgically implanted into patients. This means that it will be much harder to get approval to conduct such invasive implantations in patients than the straightforward injections which were conducted in the first wave of studies, but most of us who have now moved towards a blood vessel engineering approach feel that there is a greater likelihood of long-term success even if it may take a decade or longer till we obtain our first definitive clinical results.

The second conceptual shift which has occurred in this field is the realization that blood vessel engineering is not only important for treating patients with blockages in their arteries. In fact, blood vessel engineering is critical for all forms of tissue and organ engineering. In the US, more than 120,000 people are awaiting an organ transplant but only a quarter of them will receive an organ in any given year. The number of people in need of a transplant will continue to grow but the supply of organs is limited and many patients will unfortunately die while waiting for an organ which they desperately need. The advances in stem cell biology have made it possible to envision creating organs or organoids (functional smaller parts of an organ) which could help alleviate the need for organs. One thing that most organs and tissues need is a network of tiny blood vessels that permeate the whole tissue: small capillary networks. For example, a liver built out of liver cells could never function without a network of tiny blood vessels which supply the liver cells with metabolites and oxygen. From an organ engineering point of view, microvessel engineering is just as important as the building of functional arteries.

In one of our recent projects, we engineered functional human blood vessels by combining bone marrow derived stem cells with endothelial cells (the cells which coat the inside of all blood vessels). It turns out that stem cells do not become endothelial cells but instead release a molecular signal – the protein SLIT3- which instructs the endothelial cells to assemble into networks. Using a high resolution microscope, we watched this process in real-time over a course of 72 hours in the laboratory and could observe how the endothelial cells began lining up into tube-like structures in the presence of the bone marrow stem cells. The human endothelial cells were like building blocks, the human bone marrow stem cells were the builders “overseeing” the construction. When we implanted the assembled blood vessel structures into mice, we could see that they were fully functional, allowing mouse blood to travel through them without leaking or causing any other major problems (see image, taken from reference 3).

I am sure that SLIT3 is just one of many molecular cues released by the stem cells to assemble functional networks and there are many additional mechanisms which still need to be discovered. We still need to learn much more about which “builders” and which “building blocks” are best suited for each type of blood vessel that we want to construct. The fact that human fat tissue can serve as an important resource for obtaining adult stem cells(“builders”) is quite encouraging, but we still know very little about the overall longevity of the engineered vessels, the best way to implant them into patients, and the key molecular and biomechanical mechanisms which will be required to engineer organs with functional blood vessels. It will be quite some time until the first fully engineered organs will be implanted in humans, but the dizzying rate of progress suggests that we can be quite optimistic.

Two scientific papers that were published in the journal Nature in the year 2000 marked the beginning of engineering biological circuits in cells. The paper “Construction of a genetic toggle switch in Escherichia coli” by Timothy Gardner, Charles Cantor and James Collins created a genetic toggle switch by simultaneously introducing an artificial DNA plasmid into a bacterial cell. This DNA plasmid contained two promoters (DNA sequences which regulate the expression of genes) and two repressors (genes that encode for proteins which suppress the expression of genes) as well as a gene encoding for green fluorescent protein that served as a read-out for the system. The repressors used were sensitive to either selected chemicals or temperature. In one of the experiments, the system was turned ON by adding the chemical IPTG (a modified sugar) and nearly all the cells became green fluorescent within five to six hours. Upon raising the temperature to activate the temperature-sensitive repressor, the cells began losing their green fluorescence within an hour and returned to the OFF state. Many labs had used chemical or temperature switches to turn gene expression on in the past, but this paper was the first to assemble multiple genes together and construct a switch which allowed switching cells back and forth between stable ON and OFF states.

The same issue of Nature contained a second land-mark paper which also described the engineering of gene circuits. The researchers Michael Elowitz and Stanislas Leibler describe the generation of an engineered gene oscillator in their article “A synthetic oscillatory network of transcriptional regulators“. By introducing three repressor genes which constituted a negative feedback loop and a green fluorescent protein as a marker of the oscillation, the researchers created a molecular clock in bacteria with an oscillation period of roughly 150 minutes. The genes and proteins encoded by the genes were not part of any natural biological clock and none of them would have oscillated if they had been introduced into the bacteria on their own. The beauty of the design lay in the combination of three serially repressing genes and the periodicity of this engineered clock reflected the half-life of the protein encoded by each gene as well as the time it took for the protein to act on the subsequent member of the gene loop.

Both papers described the introduction of plasmids encoding for multiple genes into bacteria but this itself was not novel. In fact, this has been a routine practice since the 1970s for many molecular biology laboratories. The panache of the work lay in the construction of functional biological modules consisting of multiple genes which interacted with each other in a controlled and predictable manner. Since the publication of these two articles, hundreds of scientific papers have been published which describe even more intricate engineered gene circuits. These newer studies take advantage of the large number of molecular tools that have become available to query the genome as well as newer DNA plasmids which encode for novel biosensors and regulators.

Synthetic biology is an area of science devoted to engineering novel biological circuits, devices, systems, genomes or even whole organisms. This rather broad description of what “synthetic biology” encompasses reflects the multidisciplinary nature of this field which integrates ideas derived from biology, engineering, chemistry and mathematical modeling as well as a vast arsenal of experimental tools developed in each of these disciplines. Specific examples of “synthetic biology” include the engineering of microbial organisms that are able to mass produce fuels or other valuable raw materials, synthesizing large chunks of DNA to replace whole chromosomes or even the complete genome in certain cells, assembling synthetic cells or introducing groups of genes into cells so that these genes can form functional circuits by interacting with each other. Synthesis in the context of synthetic biology can signify the engineering of artificial genes or biological systems that do not exist in nature (i.e. synthetic = artificial or unnatural), but synthesis can also stand for integration and composition, a meaning which is closer to the Greek origin of the word. It is this latter aspect of synthetic biology which makes it an attractive area for basic scientists who are trying to understand the complexity of biological organisms. Instead of the traditional molecular biology focus on studying just one single gene and its function, synthetic biology is engineering biological composites that consist of multiple genes and regulatory elements of each gene. This enables scientists to interrogate the interactions of these genes, their regulatory elements and the proteins encoded by the genes with each other. Synthesis serves as a path to analysis.

One goal of synthetic biologists is to create complex circuits in cells to facilitate biocomputing, building biological computers that are as powerful or even more powerful that traditional computers. While such gene circuits and cells that have been engineered have some degree of memory and computing power, they are no match for the comparatively gigantic computing power of even small digital computers. Nevertheless, we have to keep in mind that the field is very young and advances are progressing at a rapid pace.

One of the major recent advances in synthetic biology occurred in 2013 when an MIT research team led by Rahul Sarpeshkar and Timothy Lu at MIT created analog computing circuits in cells. Most synthetic biology groups that engineer gene circuits in cells to create biological computers have taken their cues from contemporary computer technology. Nearly all of the computers we use are digital computers, which process data using discrete values such as 0’s and 1’s. Analog data processing on the other hand uses a continuous range of values instead of 0’s and 1’s. Digital computers have supplanted analog computing in nearly all areas of life because they are easy to program, highly efficient and process analog signals by converting them into digital data. Nature, on the other hand, processes data and information using both analog and digital approaches. Some biological states are indeed discrete, such as heart cells which are electrically depolarized and then repolarized in periodical intervals in order to keep the heart beating. Such discrete states of cells (polarized / depolarized) can be modeled using the ON and OFF states in the biological circuit described earlier. However, many biological processes, such as inflammation, occur on a continuous scale. Cells do not just exist in uninflamed and inflamed states; instead there is a continuum of inflammation from minimal inflammatory activation of cells to massive inflammation. Environmental signals that are critical for cell behavior such as temperature, tension or shear stress occur on a continuous scale and there is little evidence to indicate that cells convert these analog signals into digital data.

Most of the attempts to create synthetic gene circuits and study information processing in cells have been based on a digital computing paradigm. Sarpeshkar and Lu instead wondered whether one could construct analog computation circuits and take advantage of the analog information processing systems that may be intrinsic to cells. The researchers created an analog synthetic gene circuit using only three proteins that regulate gene expression and the fluorescent protein mCherry as a read-out. This synthetic circuit was able to perform additions or ratiometric calculations in which the cumulative fluorescence of the mCherry was either the sum or the ratio of selected chemical input concentrations. Constructing a digital circuit with similar computational power would have required a much larger number of components.

The design of analog gene circuits represents a major turning point in synthetic biology and will likely spark a wave of new research which combines analog and digital computing when trying to engineer biological computers. In our day-to-day lives, analog computers have become more-or-less obsolete. However, the recent call for unconventional computing research by the US Defense Advanced Research Projects Agency (DARPA) is seen by some as one indicator of a possible paradigm shift towards re-examining the value of analog computing. If other synthetic biology groups can replicate the work of Sarpeshkar and Lu and construct even more powerful analog or analog-digital hybrid circuits, then the renaissance of analog computing could be driven by biology. It is difficult to make any predictions regarding the construction of biological computing machines which rival or surpass the computing power of contemporary digital computers. What we can say is that synthetic biology is becoming one of the most exciting areas of research that will provide amazing insights into the complexity of biological systems and may provide a path to revolutionize biotechnology.

We recently published a PLOS ONE paper (Mitochondrial respiration regulates adipogenic differentiation of human mesenchymal stem cells) in which we studied how the metabolism of an adult stem cell can influence its ability to differentiate. Human bone marrow mesenchymal stem cells (also known as marrow stromal cells, marrow progenitor cells or MSCs) can be converted into fat (adipocytes), cartilage (chondrocytes) or bone (osteoblasts). The work performed by Yanmin Zhang and Glenn Marsboom in my lab showed that MSCs undergo a major metabolic shift towards increased mitochondrial oxidation when they become fat cells and that suppressing mitochondrial respiration can prevent their differentiation. The metabolic state of the adult stem cells is therefore not only an indicator of their “stemness”, it can be used to either promote or suppress their differentiation.

Dr. Peter Toth, one of the co-authors on the paper, helped us acquire some really beautiful images of the cells that I would like to share with the readers of the blog. The image below shows undifferentiated adult human bone marrow mesenchymal stem cells (MSCs) that were exposed to an adipogenic differentiation medium, i.e a combination of factors which induces the formation of fat cells (adipocytes). However, as with many stem cell differentiation protocols, not all stem cells turned into fat cells. The cells on the right have a typical fat-like structure in which cells are full of round lipid droplets. The neighboring cells on the left are MSCs that have not (yet?) become fat cells. We stained the cells with the fluorescent mitochondrial dye JC-1. Depolarized mitochondria appear green and hyperpolarized mitochondria red. As you can see, the cells on the left have a much higher mitochondrial membrane potential (significant amount of red among the green mitochondria) than their fat neighbors on the right (mostly green mitochondria, all of them located between lipid droplets). By capturing both cell types next to each other, we could show an illustrative example of how entwined metabolism and stem cell differentiation are. The morphology and metabolic state of neighboring cells in this image were quite different, despite the fact that all cells were subjected to the same cocktail of differentiation factors. The blue-appearing dye is DAPI and stains nuclei of cells so one can tell the cells apart. Each cell in this image has one blue nucleus.

The image was published with a PLoS ONE CC-BY license. Feel free to use it as an example of adult stem cell differentiation or how mitochondrial morphology and function can vary between stem cell and its differentiated progeny, as long as you attribute the original PLoS One paper. The image in the paper also has a scale bar and asterisks/arrows pointing out the specific cells.

I recently used the Web of Science database to generate a list of the most highly cited papers in stem cell research. As of July 2013, the search for original research articles which use the key word “stem cells” resulted in the following list of the ten most widely cited papers to date:

Three of the articles (Donehower et al, Al-Hajj et al and Lu et al) in this “top ten list” do not focus on stem cells but are actually cancer research papers. They were probably identified by the search because the authors may have made comparisons to stem cells or used stem cells as tools.The remaining seven articles are indeed widely known in the stem cell field.

The Science paper by Pittenger and colleagues in 1999 provided a very comprehensive description of mesenchymal stem cells (MSCs), a type of adult stem cell which is found in the bone marrow alongside hematopoietic stem cells (HSCs). Despite the fact that MSCs and HSCs are both adult stem cells in the bone marrow, they have very different functions. HSCs give rise to circulating blood cells, whereas MSCs primarily form bone, fat and cartilage as was nicely demonstrated by Pittenger and colleagues.

The article by Thomson and colleagues was published in 1998 in the journal Science described the derivation of human embryonic stem cells (ESCs) and revolutionized the field of stem cell research. While adult stem cells have a very limited capacity in terms of lineages they can turn into, ESCs are derived from the early blastocyst stage of embryonic development (within the first 1-2 weeks following fertilization) and thus retain the capacity to turn into a very wide range of tissues, such as neurons, heart cells, blood vessel cells or liver cells. This paper not only identified the methods for isolating human ESCs, but also how to keep them in culture and expand them as undifferentiated stem cells.

The Cell paper by Takahashi and Yamanaka in 2006 represented another major advancement in the field of stem cell biology, because it showed for the first time that a mouse adult skin cell (fibroblast) could be reprogrammed and converted into a truly pluripotent stem cell (an induced pluripotent stem cell or iPSC) which exhibited all the major characteristics of an embryonic stem cell (ESC). It was as if the adult skin cell was traveling back in time, erasing its identity of having been a skin cell and returning to primordial, embryonic-like stem cell. Only one year later, Dr. Yamanaka’s group was able to demonstrate the same phenomena for adult human skin cells in the 2007 Cell paper (Takahashi et al), and in the same year a different group independently confirmed that adult human cells could be reprogrammed to the iPSC state (Science paper by Yu et al in 2007). The generation of iPSCs described in these three papers is probably the most remarkable discovery in stem cell biology during the past decade. It is no wonder that each of these three papers have been cited several thousand times even though they were published only six or seven years ago, and that Dr. Yamanaka was awarded the 2012 Nobel prize for this pioneering work.

All five of the above-mentioned stem cell papers have one thing in common: the results have been repeated and confirmed by numerous independent laboratories all over the world. However, this does not necessarily hold true for the other two highly cited stem cell papers on this list.

The 2002 Nature paper by Jiang and colleagues from Dr. Verfaillie’s laboratory at the University of Minnesota proposed that the bone marrow contained a rather special subset of adult MSCs which had a much broader differentiation potential than had been previously recognized. While adult MSCs were thought to primarily turn into bone, cartilage or fat when given the appropriate cues, this rare new cell type – referred to as MAPCs (multipotent adult progenitor cells) – appeared to differentiate into a much broader range of tissues. The paper even showed data from an experiment in which these adult mouse bone marrow stem cells were combined with embryonic cells and gave rise to a chimeric mouse. i.e. a mouse in which the tissues were in part derived from standard embryonic cells and in part from the newly discovered adult MAPCs. Such chimerism suggested that the MAPCs were embryonic-like, contributing to the formation of all the tissues in the mice. At the time of its publication, this paper was met with great enthusiasm because it proved that the adult body contained embryonic-like cells, hidden away in the bone marrow, and that these MAPCs could be used to regenerate ailing organs and tissues without having to use ethically problematic human embryonic stem cells.

There was just one major catch. Many laboratories around the world tried to replicate the results and were unable to identify the MAPCs, and even when they found cells that were MAPCs, they were unable to confirm the embryonic-like nature of the cells. In a remarkable example of investigative journalism, the science journalists Peter Aldhous and Eugenie Reich identified multiple irregularities in the publications involving MAPCs and documented the inability of researchers to replicate the findings by publishing the results of their investigation in the New Scientist (PDF).

The second high profile stem cell paper which was also plagued by an inability to replicate the results was the 2001 Nature paper by Orlic and colleagues. In this paper from Dr. Anversa’s laboratory, the authors suggested that adult hematopoietic (blood-forming) stem cells from the bone marrow could regenerate an infarcted heart by becoming heart cells (cardiomyocytes). It was a rather bold claim, because simply injecting these blood-forming stem cells into the heart seemed to be sufficient to redirect their fate. Instead of giving rise to red and white blood cells, these bone marrow cells were generating functional heart cells. If this were the case, then every patient could be potentially treated with their own bone marrow and grow back damaged heart tissue after a heart attack. Unfortunately, it was too good to be true. Two leading stem cell laboratories partnered up to confirm the results, but even after years of experiments, they were unable to find any evidence of adult bone marrow stem cells converting into functional heart cells. They published their findings three years later, also in the journal Nature:

Interestingly, the original paper which had made the claim that bone marrow cells can become functional heart cells has been cited nearly 3,000 times, whereas the refutation by Murry and colleagues, published in the same high-profile journal has been cited only 1,150 times. The vast majority of the nearly 3,000 citations of the 2001 paper by Orlic and colleagues occurred after it had been refuted in 2004! The 2001 Orlic et al paper has even been used to justify clinical trials in which bone marrow was obtained from heart attack patients and injected into their hearts. As expected after the refutation by Murry and colleagues, the success of these clinical trials was rather limited One of the largest bone marrow infusion trials in heart attack patients was recently published, showing no success of the therapy.

These claims of the two papers (Orlic et al and Jiang et al) were quite innovative and exciting, and they were also published in a high-profile, peer-reviewed journal, just like the other five stem cell papers. The crucial difference was the fact that their findings could not be replicated by other laboratories. Despite their lack of replicability, both papers had an enormous impact on the field of stem cell research. Senior scientists, postdocs and graduate students may have devoted a substantial amount of time and resources to developing projects that built on the findings of these two papers, only to find out that they could not be replicated. If there is a lesson to be learned, it is that we need to be rather cautious in terms of our enthusiasm for new claims in stem cell biology until they have been appropriately confirmed by other researchers. Furthermore, we need to streamline the replicability testing process so that we do not have to wait years before we find out that one of the most highly prized discoveries cannot be independently confirmed.

Update 7/24/2013: Peter Aldhous reminded me that the superb job of investigative journalism into the question of MAPCs was performed in partnership with the science writer Eugenie Reich, the author of a book on scientific fraud. I have updated the blog post to reflect this.